Control of nucleotide homeostasis and genomic integrity in Trypanosoma bruceirole of hd nucleotidases and base excision repair

Abstract

The first aim of this research was the identification of HsSAMHD1 orthologues in T. brucei, as well as the evaluation of their role in cell viability and homeostasis of pyrimidine dNTPs. To this purpose, the following specific objectives were proposed: 1. Identify potential HsSAMHD1 orthologues in T. brucei with a predicted dNTPase activity and determine their intracellular localization. 2. Evaluate the role of HsSAMHD1 orthologues in cell viability and cell cycle progression. 3. Establish the role of the identified dNTPases in the supply of pyrimidine and purine nucleosides. 4. Analyze the contribution of dNTPases to pyrimidine and thymidylate biosynthesis. 5. Determine the impact of perturbations in dNTP hydrolysis on genomic integrity. DNA lesions can derive from polymerase action during replication or external agents that modify DNA bases, which can lead to severe damage, such as mutations, DNA breaks or AP sites. In the case of T. brucei, during infection parasites are especially exposed to an intense oxidative stress as a result of the response of the immune system. In order to counteract this situation and sustain genomic integrity, cells trigger multiple repair mechanisms. Previous studies conducted in the lab have already demonstrated the importance of UNG for T. brucei infectivity, as UNG-deficient parasites showed reduced virulence. Thus, a second general aim of this thesis was to analyze the occurrence of oxidative stress and the activation of DNA repair pathways during host-pathogen interactions in vivo. The following specific objectives were proposed: 1. Evaluate the impact of oxidative stress on DNA integrity in T. brucei BSFs exposed to genotoxic compounds in vitro. 2. Analyze the consequences on parasites of the potential oxidative stress that arises upon murine infection and the activation of a DNA damage response. 3. Determine the relevance of UNG and the BER pathway in counteracting DNA damage produced by oxidative stress.